Magnetite-Quantum Dot Immunoarray for Plasmon-Coupled-Fluorescence Imaging of Blood Insulin and Glycated Hemoglobin

Abstract

New microarray chip strategies that are sensitive and selective and that can measure low levels of important biomarkers directly in a blood sample are significant for improving human health by allowing timely diagnosis of an abnormal condition. Herein, we designed an antibody-aptamer immunoarray chip to demonstrate simultaneous measurement of blood insulin and glycated hemoglobin (HbA1c) levels relevant to diabetic and prediabetic disorders using a surface plasmon microarray with validation by fluorescence imaging. To accomplish both surface plasmon and fluorescence imaging on the same sample, we decorated magnetite nanoparticles with quantum dots for covalent immobilization of aptamers for subsequent capture and isolation of the aptamers specific for insulin and HbA1c markers from 20-times diluted whole blood samples. Direct clinically relevant analysis, along with fluorescent imaging of the two markers, was achieved by this new immunoarray platform. The limit of detection was 4 pM for insulin and 1% for HbA1c. Examination of cross-talk using thrombin and platelet-derived growth factor confirmed that the designed immunoarray was highly selective for insulin and HbA1c. Surface plasmon kinetic analysis provided apparent binding constants of 0.24 (±0.08) nM and 37 (±3) μM, respectively, for the binding of insulin and HbA1c onto their surface immobilized monoclonal antibodies. Thus, quantitative imaging of ultralow levels of blood biomarker levels with binding kinetics is uniquely obtained in the designed immunoarray chip. In conclusion, this report demonstrates considerable significance of the developed magnetite-quantum dot-bioconjugate strategy for clinical diagnostics of whole blood biomarkers with characterization of molecular binding interactions.

Keywords: binding kinetics; magnetite-quantum dot; microarray; nonglucose markers; plasmon-coupled-fluorescence; whole blood analysis.

Figure 1

Assay strategy used for measuring the clinical levels of insulin and HbA1c in 20-times diluted whole blood in PBS.

Figure 2

Reflectivity changes during stepwise SPR microarray fabrication with (a) glutaraldehyde, (b) Ab_insulin or Ab_HbA1c, and (c) 0.1% BSA. The broken lines indicate the onset of the washing step (duration of 5 min).

Figure 3

Difference images, line profiles, and sensograms of (A) control, (B) 50 pM and 6%, and (C) 500 pM and 10% insulin and HbA1c levels, respectively, in whole blood.

Figure 4

SPR sensograms for (A) insulin and (B) HbA1c and calibration plots for (C) insulin and (D) HbA1c detection in 20-times diluted whole blood (N = 3 replicates).

Figure 5

Difference images, line profiles, and sensograms of (A) T1D and (B) T2D whole blood patient samples.

Figure 6

Fluorescence images of aptamer control (A,B), 20-times diluted whole blood conjugates with residual insulin and HbA1c (C,D), MNP-QD-aptamer captured 50 pM insulin and 6% HbA1c (E,F), and 500 pM insulin and 10% HbA1c (G,H) in 20-times diluted whole blood upon binding to their respective surface immobilized antibodies on the SPR microarray chip. Scale bar: 20 μm for (A–H).